1. Field of the Invention
The present invention relates to a novel protein or polypeptide modified in bonding with a polyethylene glycol derivative, which is useful as a physiologically active or medically active substance or an intermediate compound therefor, and a method for producing the same. The present invention also relates a reactive polyethylene glycol derivative which is an intermediate compound for the novel protein or polypeptide above-mentioned.
2. Description of the Related Art
Many protein, polyaminoacid and peptide substances having a physiological or medical activity have recently been discovered, permitting expectation of more common application to medical substance. However, these protein and peptide substances have only a short half-life in blood when administered in vivo, giving a sufficient pharmacological effect in few cases. In order to utilize these substances as medical substance, therefore, it is believed to be absolutely necessary to improve behaviors in vivo by some method or other.
It is known that many of physiologically active or medically active substances administered in vivo, particularly into the blood flow, disappear from the biological body through glomerular filtration in the kidney. This glomerular filtration process may be considered a kind of molecular sieve in principle: substances of a molecular weight smaller than that of albumin (about 60,000) which is plasma protein indispensable for a biological body are excreted as a rule. In order to improve in vivo behaviors of protein and peptide medical substance disappearing from the body through glomerular filtration, therefore, it has conventionally been believed to be necessary to increase the molecular weight of medical substance through various chemical modifications.
As a method for improving behaviors in vivo of a protein or peptide medical substance, chemical modification using a water-soluble polymer typically represented by polyethylene glycol (hereinafter abbreviated as "PEG" as required) has popularly been applied. PEG has rather a long history: this substance has been utilized and studied widely in various areas since its synthesis in 1859. In the areas of biochemistry and medicines or medical drugs as well, it is confirmed that PEG exerts no interaction to protein except for physical stereo-hindrances, and no change is observed in protein CD spectrum even in a high-concentration aqueous PEG solution. This suggests that modification with PEG does not destroy the higher-order structure of protein. When, for example, PEG of a molecular weight of 4000 is administered to a dog at a rate of 90 mg/kg/day for a period of a year, no abnormality is observed in body weight or in a pathological or hemological inspection. Administration of PEG to a guinea pig does not cause an allergic symptom. Safety of PEG is thus commonly confirmed. By modifying protein with PEG having the unique properties as described above, it is expected to be possible to develop a protein or peptide medical substance having a long life in vivo, which is hard to be recognized not only by the immune system but also by the reticuloendothelial system.
When modifying a protein or peptide medical substance with PEG, the following advantages are available. When non-denatured protein is insoluble under a physiological pH condition or only partially soluble, it is possible to considerably improve solubility under the physiological pH condition by modifying protein with PEG, and also to reduce immune response of non-denatured protein. For these advantages, many types of protein or peptide medical substance have been modified with PEG to date for utilization in medical drugs regarding general remarks, see Inada, Y., Yoshimoto, T., Matsushima, A. and Saito, Y. (1986) Trends Biotechno, 1, 4: 68-73!.
PEG derivatives so far used for modifying protein or peptide medical substance include: 2-(alkoxypolyethyleneglycoxy)-4,6-dechlorotriadine Abuchowski, A., Van Es, T., Palczuk, N. C. and Davis, F. F. (1977) J. Biol. Chem. 252, 3578-3581!; 6-(alkoxypolyethyleneglycoxy)-S-carboxamide-methyl-dithiocarbonate King, T. P. and Weiner, C. (1980) Int. J. Peptide Protein Res. 16, 147-155!; 2-(alkoxypolyethylene-glycoxy)-N-succinimyzilsuccinate Abuchowski, A, Kazo, G. M., Verhoest, C., Van Es, T., Kafkewitz, D., Viau, A. and Davis, F. (1984) Cancer Biochem. Biophys. 7, 175-186!; 2-(alkoxypolyethyleneglycoxy) carboxyimidazole Beauchamp, C. O., Gonias, S. L., Menapace, D. P. and Pizzo, S. V. (1983) Anal. Biochem. 131, 25-33!; 2-alkoxypolyethyleneglycoxy)-2,4,5-trichlorobenzene Versonese, F. M., Largajolli, R., Boccu, E., Benassi, C. A. arid Schiavon, O. (1985) Applied Biochem. Biotech., 11, 141-152!; 2-(alkoxypolyethylene-glycoxy)-4-nitrobenzene Versonese, F. M., Largajolli, R., Boccu, E., Benassi, C. A. and Schiavon, O. (1985) Applied Biochem. Biotech. 11, 141-152!; 2-(alokxypolyethylene-glycoxy)-2,2,2-trifluoroethane Delgads, C., Patel, J. N., Francis, G. B. and Fisher, D. (1990) Biotech. Applied Biochem. 12, 119-128!; 2-(alkoxypolyethylenealdehyde) Andrews, B. A., Head, D. M., Dunthrone, P. and Asenjo, J. A. (1990) Biotech. Tech. 4, 49-54!; and 2-alkoxypolyethylene-glycoxymethylepoxide Andrews, B. A., Head, D. M., Dunthrone, P. and Adenjo, J. A. (1990) Biotech. Tech. 4, 49-54!.
Regarding modification with a PEG derivative, a number of patent applications have been filed to date. Some examples include, for example, Japanese Provisonal Patent Publication JP-A-61-178,926; Japanese Provisional Patent Publication JP-A-61-249,388; Japanese Provisional Patent Publication JP-A-1-316,400; Japanese Provisional Patent Publication JP-A-2-117,920; Japanese Provisional Patent Publication JP-A-3-88,822; Japanese Proisional Patent Publication JP-A-5-117,300; Japanese Provisional Patent Publication JP-A-132,431; Japanese Provisional Patent Publication JP-A-5-214,092; and Japanese Provisional Patent Publication JP-A-5-503,092. All these inventions are based on attention to the high water solubility of PEG, having therefore a terminal structure comprising a short-chain aliphatic group.
However, the method of improving behaviors in vivo by increasing the molecular weight of a protein or peptide medical substance through PEG modification is considered to suffer on the other hand limitations imposed on medical substances to which it is applicable. For example, most of conventional protein or peptide medical substances of which PEG modification has been confirmed to have improved behaviors in vivo and increased pharmacological effect are limited to enzymes. A conceivable cause is the pharmacological effect expressing mechanism unique to enzymes.
An enzyme having a physiological activity expresses its effect by causing a substance detrimental for a biological body or a substance specifically required at a site suffering from a disease such as tumor to specifically disappear through a chemical reaction such as metabolism. In the case of PEG modification of an enzyme, therefore, it is possible to increase the pharmacological effect by only causing enzyme molecules to be present in blood for a long period of time. However, such a method of increasing the molecular weight of a medical substance through PEG modification is not clearly a method commonly applicable to all protein or peptide medical substance other than enzymes.
More particularly, a protein or peptide medical substances requiring improvement of behaviors in vivo necessitates bonding with a receptor, for example, for the purpose of displaying its pharmacological effect, PEG modification cannot be expected to increase the pharmacological effect, but the pharmacological effect decreases in many cases as a result of impairment of receptor bonding caused by stress hindrance of polymer PEG used for modification. For example, in Ehrat, M, and Luisi, P. L. (1983) Biopolymer 22, 569, 573, when PEG-modifying insulins to be bonded with a receptor for more remarkable pharmacological effect, a decrease in pharmacological effect in vivo by up to 50% is reported as compared with non-modified insulin.
Therefore, for many of protein, polyaminoacid and peptide substances expected to become applicable to medical uses, bonding with a receptor or the like is a necessary prerequisite for the expression of pharmacological effect. Under such circumstances, there is an increasing demand for achievement of a novel method of improving behaviors in vivo of medical substances, which does not impair bonding with a site where a pharmacological effect is to be expressed, such as a receptor or the like, i.e., novel means for chemical modification, and a medical substance comprising a pharmacologically active substance modified by such means.